Histone Metabolism in Skeletal Muscle Differentiation

Lough, John

Abstract

This proposal describes two approaches to assess the role of histone proteins during normal and dystrophic myogenesis. These studies will help elucidate how the differentiated state of muscle is acquired and maintained: I) Histone variant metabolism during myogenesis: These experiments will utilize one- and two-dimensional gel electrophoresis to verify that the five major classes of histones--H1, H2a, H2b, H3 and H4--are synthesized in cultures of postmitotic myotubes. The extent of myotube histone synthesis will be compared to that in dividing myoblasts. Mass and synthesis ratios of variants within each major histone class will be determined with respect to proliferative and differentiative stage. The stability of de novo synthesized myotube histones, and ubiquitinated histones, will be assessed. Finally, active and inactive chromatin in myoblast nuclei will be separated using micrococcal nuclease digestion or a novel technique involving DNase I-directed nick translation with biotinylated nucleotides, followed by assessment of whether de novo synthesized histones are associated with active genes such as M-creatine kinase which will be probed by dot-blot hybridization analysis. II) Role of polyamines in modulating histone phosphorylation, cellular proliferation and differentiation: These experiments will employ intact cells and isolated nuclei to probe the relationship between intracellular polyamine levels, histone phosphorylation and cellular proliferation and differentiation. Assessment in isolated nuclei will focus on the concentration-dependent effects of cyclic nucleotides, divalent cations and polyamines (spermine, spermidine, putrescine) on phosphorylation, the effects of polyamines on histone phosphatase activity, and mixing experiments to determine whether modulations in phosphorylation are caused by changing histone kinase activity or chromatin structural changes which influence the susceptibility of H3 to phosphorylation.